Fungicides are chemical compounds commonly used to control the fungi affecting cultures mainly.
Many of these compounds are organic synthetic substances with a wide range of toxicity and are persistent in the environment. Thousands of tons of fungicides are annually applied worldwide to control various fungi.
The B. cinerea fungus is responsible for grey rot and it may attack over 200 species of cultivated plants, especially those growing in a greenhouse. This is a saprofite fungus that attacks dead or senescent tissues, penetrates the tissue and causes the plant's death, thus resulting in huge losses to farmers. The control of this fungus is performed through chemical agents. At present, benomil and iprodione are used, but will be taken from the market, since they have developed resistance in the fungus.
An additional problem with fungicides currently being used is that their concentration must be increased in order to control the pathogen, which causes problems both in the environment and in the organisms in contact with the chemical agent. Therefore, new products are required should be highly effective and have low environmental toxicity.
As an alternative to synthetic agents, botanical pesticides have the advantage of being natural compounds, and thus are safer for humans and the environment. Specifically, botanical pesticides are less toxic than conventional pesticides and they generally affect the target organism only or those closely related thereto. Also they decay rapidly in the soil, and thus are ideal components for Integrated Pests Management (IPM) programs.
Quillay (Quillaja saponaria Molina) is a tree of the Rosaceae family, native to Chile, and its biomass contains molecules called saponins, specifically of the triterpenoid type. The saponins provide the extracts of this tree with unique characteristics, which have been used for decades in the various industries, such as foodstuff and drinks, mining, agriculture, animal feeding, and treatment of effluents, among others.
The main properties of the quillay extracts are: reduction of surface tension, formation of a persistent foam, and emulsification of fats and oils. There is a great amount of scientific literature (papers and patents) that describe the use of these extracts for different industrial applications. U.S. Pat. No. 4,933,371, incorporated herein by reference, describes the use of saponins of different plants (e.g. yucca, quillay, agave, and tobacco) as acaricide.
Quillay's biocide activity has been previously described for instance in Apablaza et al (Fitopatologia, Vol. 39, No 3, 2004, pages. 144-149, ISSN 0430-6155), where experiments are described about the control of cucurbitaceae oidium [Erysiphe cichoracearum DC. ex Mérat and Sphaerotheca fuliginea (Schlecht ex Fr.) Poll] with the saponin-containing quillay extracts (Quillaja saponaria Mol.) QL 1000 and QL 30B. The experiments provided satisfactory results in cucumber and pumpkins. In ill leaves treated with these extracts, a color change of oidium was noted, from white to brown. With the aid of a magnifying glass or microscope, disorganization of the mycelium in relation to the control was noted. The purpose of this study was to prove and observe the effect of the QL 1000 extract on the mycelium and fungus conidia under a light microscope. Pumpkin plants infected with oidium were treated with three doses of the product. Treated and control leaves were taken and they were subject to sheet scraping and microscopic observation. Tissue cuts were observed. The results of the experiments, both under the sheet scraping technique and tissue cuts, showed full chains of oidium normal conidia and conidiophore in the control leaves; partially affected and shorter chains at the 50 ppm dose of saponins; fully disorganized chains and conidia at lysis at the 100 ppm dose; and short, partially disorganized chains at the 200 ppm dose of saponins. These results allowed proving the oidium control with quillay QL-100. It should be noted in this respect that BASF QL products correspond to a 100% natural quillay-based extract nematicide, especially indicated for the control of nematodes in vineyards and citric trees.
In the EP publication 1867230, incorporated herein by reference, a natural product is described recording the antifungal and growth promoting effect in order to improve the productivity of plants, which comprises at least two active substances derived from a) quillay (Quillaja saponaria), b) quinoa (Chenopodium quinoa), c) tea (Camellia spp., e.g. Camellia oliefera, Camellia sinensis, Camellia chekiangoleosa, C. drupifera, C. reticulata or C. japonica), and d) a saponin contained in a plant material other than (a), (b) or (c).
In the international patent publication WO2007/04680, incorporated herein by reference, an organic insecticide blend is described for the control of Aedes aegypti, comprising extracts from Quillaja saponaria, Chrysanthemum cinerariaefolium (pyrethrins), Azadirachta indica, Azadirachtin, and a garlic waterish extract (Allium spp).
US Patent Publication US2006/121126, incorporated herein by reference, describes a composition containing an oxygen peroxide generating compound and saponin glucoside compounds, which by being combined show an increased activity against bugs, pathogens, algae, moulds and fungal activity.
A great number of patent documents have been found, which describe the use of quillay, the taking of extracts are taken, and its applications, such as CL 625-1995 application, incorporated herein by reference, where a procedure is described to produce white-colored saponins from quillay wood; CL publication 1203-1998, incorporated herein by reference, that discloses a saponin production process using quillay wood as raw material by soaking and then pressing or laminating the pre-sized wood; CL publication 2573-2002, incorporated herein by reference, that provides a production process for high-purity quillay extract based on the use of the whole biomass, comprising chipping the wood, extracting the solids, purifying the extract, filtering the blend, and concentrating the extract at the filtering and pasteurizing stage, CL publication 2772-2002, which is equivalent to US Application Publication 2005/074508, incorporated herein by reference, that describes a product for the control of phytoparasitary nemotodes prepared on the basis of a waterish extract of quillaja saponaria Molina (quillay) containing both the saponin fraction and the non-saponin fraction; and the method to control nematodes where the product is applied in farming crops. These documents mainly aim at the use of saponins and in particular to the waterish fraction of the extract, always from natural sources and in vivo cultures.
The use of plants as the source of substances with biological activity requires biomass to be available, which does not always allow keeping the balance between the production and use, thus overexploitation situations of the product occurring. Also, the chemical composition of plants is strongly affected by the environment where they develop and grow, this being a problem when using them as a source for the production of active principles. This is why the development of methodologies is required allowing the sustainable use of natural resources.
The use of the in vitro culture technique of vegetal tissues in order to obtain active extracts is already known in the previous art.
The U.S. Pat. No. 7,160,706, incorporated herein by reference, describes the use of this technique in order to obtain secondary metabolites-enriched material. More specifically, under this patent, work is done to obtain alkaloids from Papaver sp. plants.
Although the in vitro cultivation of quillay has been described in the state of art from seeds and through somatic embryogenesis (Vega A y Prehn D (2005) “Inducción e inicio de maduración in vitro de tejido embriogénico de Quillaja saponaria”, Ciencia e Investigación Agraria 32(3):197-207), this is first report where axillary shoots (or buds) of adult trees are used.